Oral Presentation 50th International Society for the Study of the Lumbar Spine Annual Meeting 2024

Introduction: Low back pain is closely associated with intervertebral disc (IVD) degeneration. This process is characterized by extracellular matrix (ECM) degradation in the annulus fibrosus (AF), leading to substrate stiffening and disorganization of the collagen architecture ^1,2. These changes can affect cell behavior through mechanoreceptors like transient receptor potential (TRP) channels, particularly TRPV4, which is highly expressed in the AF and is linked to pain and inflammation ^3-6. However, AF cell-substrate interaction with TRPV4 remains unexplored. We hypothesize that TRPV4 activation is influenced by substrate stiffness and topography, altering calcium (Ca2+) influx and modulating inflammation and degeneration-related targets. This study aims to fabricate substrates of different stiffness and topography to test their effects on TRPV4 activation, initially in static culture and later under stretching.

Methods: Chambers with various substrate stiffness were fabricated by adjusting the mixture of polydimethylsiloxane (PDMS) Sylgard 184 and 527 (0, 14, and 24 wt%). The PDMS stiffnesses were determined through uniaxial tensile testing until rupture (n=5). The surface strain (20% stretch, 1Hz) was determined by LaVision Digital Image Correlation (n=3). Substrate biocompatibility was assessed using an alamarBlue assay with bovine AF cells (n=5). The effects of substrate stiffness on total and maximal Ca2+ influx in AF cells were explored following TRPV4 pharmacological activation (0.5 µM GSK101790A) with a Fura-2 QBT assay (n=5). Aligned and random PDMS fibers were fabricated via coaxial electrospinning. PDMS fibers were obtained after dissolving the polyvinylpyrrolidone (PVP) sheath with ethanol and validated with NMR. Moreover, fiber diameter and alignment were measured using SEM images and FIJI. GraphPad Prism was utilized to perform Shapiro-Wilk normality test and one-way ANOVA.

Result: PDMS chambers were successfully fabricated with differing stiffness (9, 63, and 240 kPa) and surface strains with suitable cell viability (>80%) (Fig. 1). Upon TRPV4 activation, AF cells cultured on higher PDMS stiffness displayed increased total and maximal Ca2+ flux levels from 9 to 240 kPa substrates, highlighting TRPV4’s mechanosensitive nature in AF cells and its potential influence on downstream targets (Fig. 2). Random (2.97±0.49 µm) and aligned (3.11±0.78 µm) PDMS fibers mimicking collagen fiber diameters were also successfully fabricated (Fig. 3).

Discussion: Our biocompatible cell-substrate model successfully mimics different degeneration stages. Increasing in vitro stiffness, as observed in vivo during degeneration, led to increased TRPV4 activation, as evidenced by enhanced Ca2+ flux. These stiffness-dependent changes in intracellular Ca2+, mediated by TRPV4 activation,  modulates cell behavior; therefore, identifying TRPV4 as an interesting receptor to investigate. To improve the cell-substrate interaction model to study IVD degeneration, incorporating topography to mimic healthy (aligned) and degenerated (random) AFs is highly significant. Ongoing experiments are identifying relevant ECM, inflammatory, and degeneration-associated downstream targets. In the future, experiments will also investigate stiffness and topography in combination with the mechanical activation of TRPV4 through cyclic stretching. Overall, better understanding of AF cell-substrate interaction and the mechanotransduction process may contribute to developing new tissue engineering models and novel TRP-based therapeutics.